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Preparation, characterization and in vitro angiogenic capacity of cobaltsubstituted b-tricalcium phosphate ceramics

Meili Zhang,ab Chengtie Wu,ab Haiyan Li,c Jones Yuen,b Jiang Chang*a and Yin Xiao*b

Received 6th July 2012, Accepted 29th August 2012

DOI: 10.1039/c2jm34395a

Divalent cobalt ions (Co2+) have been shown to possess the capacity to induce angiogenesis by

activating hypoxia inducible factor-1a (HIF-1a) and subsequently inducing the production of vascular

endothelial growth factor (VEGF). However, there are few reports about Co-containing biomaterials

for inducing in vitro angiogenesis. The aim of the present work was to prepare Co-containing b-

tricalcium phosphate (Co-TCP) ceramics with different contents of calcium substituted by cobalt (0, 2,

5 mol%) and to investigate the effect of Co substitution on their physicochemical and biological

properties. Co-TCP powders were synthesized by a chemistry precipitation method and Co-TCP

ceramics were prepared by sintering the powder compacts. The effect of Co substitution on phase

transition and the sintering property of the b-TCP ceramics was investigated. The proliferation and

VEGF expression of human bone marrow mesenchymal stem cells (HBMSCs) cultured with both

powder extracts and ceramic discs of Co-TCP was further evaluated. The in vitro angiogenesis was

evaluated by the tube-like structure formation of human umbilical vein endothelial cells (HUVECs)

cultured on ECMatrix� in the presence of powder extracts. The results showed that Co substitution

suppressed the phase transition from b- to a-TCP. Both the powder extracts and ceramic discs of Co-

TCP had generally good cytocompatibility to support HBMSC growth. Importantly, the incorporation

of Co into b-TCP greatly stimulated VEGF expression of HBMSCs and Co-TCP showed a significant

enhancement of network structure formation of HUVECs compared with pure TCP. Our results

suggested that the incorporation of Co into bioceramics is a potential viable way to enhance angiogenic

properties of biomaterials. Co-TCP bioceramics may be used for bone tissue regeneration with

improved angiogenic capacity.

1 Introduction

The reconstruction of large skeletal defects remains a major

orthopaedic challenge. Autologous bone grafting is considered

as the gold standard, but insufficient donor tissue, coupled with

concerns about donor site morbidity, has limited this approach

in large-scale applications.1,2 Allogenic bone grafts can eliminate

the limitations associated with the harvesting of the autograft for

bone grafting; however, they have a high risk of inciting immu-

nological reactions and transmitting diseases in the recipients,

which impose a major restriction on the clinical application of

these treatments.1,3 Synthetic bone graft substitutes offer a

aState Key Laboratory of High Performance Ceramics and SuperfineMicrostructure, Shanghai Institute of Ceramics, Chinese Academy ofSciences, 1295 Dingxi Road, Shanghai 200050, PR China. E-mail:jchang@mail.sic.ac.cn; Fax: +86 21 52413903; Tel: +86 21 52412810bInstitute of Health and Biomedical Innovation, Queensland University ofTechnology, 60 Musk Ave, Kelvin Grove, Brisbane, QLD 4059,Australia. E-mail: yin.xiao@qut.edu.au; Fax: +61 7 31386030; Tel: +617 31386240cBiomedical Engineering School, Med-X Research Institute, ShanghaiJiaotong University, 1954 Huashan Road, Shanghai 200030, PR China

21686 | J. Mater. Chem., 2012, 22, 21686–21694

promising alternative strategy for healing severe bone injuries,

which have the advantages of osteoconductivity, unlimited

supply, long shelf life, no risk of disease/virus transfer, and

availability in various shapes, porosities and compositions.3–5

However, angiogenesis is a challenging issue for synthetic bone

grafts when used for repairing large bone tissue defects. Several

approaches are under investigation to enhance the angiogenic

property of biomaterials, such as the co-culture of endothelial

cells with other cell types on the materials, pre-vascularization of

matrices prior to cell seeding, and integrating the growth factors,

such as basic fibroblast growth factor (bFGF) and vascular

endothelial growth factor (VEGF), into biomaterial

constructs.6–9 Generally, these methods are complex and the

usage of growth factors has some disadvantages, such as high

cost, short half-life, instability and safety problems with

recombinant proteins.10 Therefore, it is of great importance to

create novel biomaterials with improved angiogenic capacity.

Cobalt (Co) is an essential trace element in human physiology

and is an integral part of vitamin B12. Co acts as a cofactor of

many metalloproteins in the body.11,12 Co2+ ions have been used

extensively to mimic a hypoxic environment in vitro, which can

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stabilize the hypoxia inducible factor-1a (HIF-1a) and subse-

quently activates HIF-1a target genes such as VEGF.13,14 It is

indicated that Co2+ ions can be used as an inorganic angiogenic

factor, which would have lower cost, higher stability, and of

potentially greater safety compared with recombinant proteins

or genetic engineering approaches. It has been recently reported

that mesoporous bioactive glass scaffolds with controllable

Co2+ ions release could enhance HIF-1a expression, VEGF

protein secretion, and bone-related genes expression in bone

marrow mesenchymal stem cells (BMSCs).15 b-Tricalcium

phosphate (b-Ca3(PO4)2, b-TCP) ceramics, typical representa-

tives of resorbable calcium orthophosphates, have been widely

studied and clinically used for bone repair applications due to

their chemical similarity to the inorganic phase of natural bone,

excellent osteoconductivity and good degradability.16 It is

speculated that the incorporation of Co into b-TCP ceramics

may be an efficient way to improve the angiogenesis for bone

repair applications.

Therefore, in the present study Co-TCP ceramics with

different contents of Co were prepared. The effect of different Co

substitution in the b-TCP ceramics on their physical properties

(phase transition and sintering property), cytocompatibility and

VEGF expression of human bone marrow mesenchymal stem

cells (HBMSCs) was investigated. In addition, angiogenesis

involves a series of complicated steps including endothelial cell

(EC) proliferation, migration, network formation and so on.17

Hereby, the in vitro angiogenesis was further evaluated by

checking the capillary-like network formation of human umbil-

ical vein endothelial cells (HUVECs) cultured on ECMatrix� in

the presence of Co-TCP powder extracts.

2 Materials and methods

2.1 Synthesis and characterization of Co-TCP powders

Co-TCP with different substitution contents of Co for Ca (2 and

5 mol%) were synthesized by a chemical precipitation method

using Ca(NO3)2$4H2O, (NH4)2HPO4 and CoCl2 as starting

materials (analytical grade, Sigma-Aldrich). The corresponding

chemical formulation was (Co0.0xCa1�0.0x)3(PO4)2, in which x%

¼ 2, 5 mol% for Co/(Ca + Co). In brief, the samples were named

as Co2-TCP and Co5-TCP, respectively. The solution containing

0.5 M of Ca(NO3)2 and CoCl2 in the designed molar ratio was

added dropwise to a 0.5 M (NH4)2HPO4 solution with stirring to

produce the target (Ca + Co)/P ratio of 1.5 (stoichiometric for

TCP). The pH of the solution was maintained between 7.5 and 8

during precipitation by the addition of ammonia. After precipi-

tation, the solution was aged overnight and then washed

sequentially in distilled water and anhydrous ethanol, dried at

60 �C for 24 h and finally calcined at 800 �C for 3 h.

The phase composition of the calcined powders was identified

by X-ray diffraction (XRD, X’pert PRO) with a monochromated

CuKa radiation and the morphology of the powders was

observed by scanning electron microscopy (SEM, FEI Company,

USA). The ratio of Ca and Co elements in the prepared materials

was quantified by electron probe microanalysis with an energy-

dispersive spectrometer (EDS) on the electron probe micro-

analyser (JXA-840A, JEOL, Tokyo). The EDS instrument was

carefully calibrated prior to EDS analysis for the samples and the

This journal is ª The Royal Society of Chemistry 2012

tested elements were quantified according to the standard data-

base in the EDS system for all known elements.

2.2 Preparation and characterization of the Co-TCP ceramics

To prepare the ceramic discs, the synthesized Co-TCP powders

were uniaxially pressed at 10 MPa in a mould of F10, using 6%

polyvinyl alcohol (PVA-124) as a binder. The green compacts

were subsequently sintered in air at 1050 and 1100 �C for 3 h,

respectively, with a heating rate of 2 �C min�1.

The crystal phase of the sintered samples was characterized by

XRD and the surface microstructure was observed by SEM. The

bulk density was measured by the Archimedes principle

according to ASTM C-20. The density was calculated as: rv ¼m1/(m2 � m3), where m1 is the dry weight in air, m2 is the satu-

rated weight, and m3 is the submerged weight in water.

2.3 Cell culture

Human bone marrow mesenchymal stem cells (HBMSCs) were

isolated and cultured based on protocols developed in previous

studies.18,19 Briefly, bone marrow was obtained from patients

undergoing hip and knee replacement surgery with informed

consent given by all donors. Mononuclear cells (MNCs) were

isolated from the bone marrow by density gradient centrifuga-

tion over Lymphoprep (Axis-Shield PoC AS, Oslo, Norway)

according to the manufacturer’s protocol. The harvested MNCs

were placed into the tissue culture flasks containing Dulbecco’s

Modified Eagle Medium (DMEM; Invitrogen Pty Ltd.) supple-

mented with 10% (v/v) fetal calf serum (FCS; In Vitro Technol-

ogies) and 1% (v/v) penicillin/streptomycin (Invitrogen) and

incubated at 37 �C in a humidified atmosphere of 95% air and 5%

CO2. The culture medium was refreshed every 3 days until the

primary mesenchymal cells reached confluency. The unattached

hematopoietic cells were removed through medium change. The

confluent cells were routinely subcultured by trypsinization. Only

early passages (p2–5) of cells were used in this study.

Human umbilical vein endothelial cells (HUVECs) were iso-

lated according to the procedures described previously.20,21

Briefly, the cord was collected from the placenta soon after birth

and stored in a sterile container filled with cord buffer (0.14 M

NaCl, 0.004 M KCl, 0.001 M phosphate buffer, pH 7.4, 0.011 M

glucose) at 4 �C until processing. A blunt 14-gauge needle was

inserted into the umbilical vein, and the needle was secured by

clamping the cord over the needle. The vein was then washed

with cord buffer perfused by a syringe through a filter until the

outlet is clear without blood. The other end of the umbilical vein

was then cannulated with the same needle as above and secured

with a clamp. 5 mL of 1 mg mL�1 collagenase (Roche Diag-

nostics, Mannheim, Germany) in cord buffer were then infused

into the umbilical vein with a syringe, and the umbilical cord was

incubated in cord buffer at 37 �C for 15 min with suspension of

the ends. After incubation, the collagenase solution containing

the ECs was flushed from the cord by perfusion with 30 mL of

cord buffer. The effluent was collected in a sterile conical

centrifuge tube and centrifuged at 1000 rpm for 10 min. After the

supernatant was discarded, the cell pellets were redispersed in

M199 (Invitrogen) supplemented with 10% (vol/vol) FBS and 1%

(vol/vol) ECs growth supplement/heparin kit (Promocell).

J. Mater. Chem., 2012, 22, 21686–21694 | 21687

Table 1 Primer sequences for the genes observed in this study

Gene Primer sequences

VEGF Fwd. 50-ATC TTT GGT CTG GCC CCC ATG-30Rev. 50-AGT CCA CCA TGG AGA CAT TCT CTC-30

18S Fwd. 50-TCG GAA CTG AGG CCA TGA TTA AG-30Rev. 50-TCT TCG AAC CTC CGA CTT TCG-30

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2.4 HBMSC proliferation in Co-TCP powder extracts

The method was carried out with the powder extracts in contact

with cells based on the standard ISO/EN 10993-5.22 The powder

extracts were prepared by soaking powders in serum-free

DMEM at a solid/liquid ratio of 200 mg mL�1. After incubation

at 37 �C for 24 h, the mixture was centrifuged and the superna-

tant was collected and sterilized by filtration through 0.2 mm filter

membranes. The levels of Ca, P and Co in the extracts were

measured using an inductively coupled plasma optical emission

spectrometer (ICP-OES, Vista-MPX, Varian, Inc., USA).

HBMSCs were seeded on a 96-well tissue culture plate at a

density of 3000 cells per well. After 24 h of incubation, the culture

medium was removed and replaced by 100 mL of test material

extracts containing 10% FCS. The DMEM without extract

supplemented with 10% FCS was used as the blank control

(Blank). At day 1 and 7, a MTT [3-(4,5-dimethylthiazol-2-yl)-

2,5-diphenyl tetrazolium bromide] assay was employed to test the

cell viability. Briefly, 10 mL of 5 mg mL�1 MTT solution was

added into each well. After incubation for a further 4 h, the

DMEM-MTT solution was removed and replaced with 100 mL

of dimethyl sulfoxide (DMSO). The absorbance was read at the

wavelength of 495 nm using a microplate spectrophotometer

(Benchmark Plus, Bio-Rad).

2.5 HBMSC proliferation on Co-TCP ceramic discs

HBMSCs were seeded on the ceramic discs in a 48-well plate at a

density of 5 � 103 cells per cm2. To avoid cell leakage during cell

seeding, first the concentrated HBMSCs suspension was dropped

onto the discs and cultured for 1 h to allow the cells to attach to

the disc surface. Then the medium was supplemented to 250 mL

per well. At pre-determined time points of 1 day and 7 days,

25 mL of 5 mg mL�1 MTT solution was added into each well.

After additional culture for 4 h, the DMEM-MTT solution was

removed and replaced with 200 mL of DMSO. 100 mL of the

reacted reagent from each well was transferred to a 96-well plate

and the absorbance was read at 495 nm using the microplate

reader.

For cell morphology observations, the cells cultured on the

ceramics for 24 h were fixed in 3% glutaraldehyde buffered in

0.1 M of sodium cacodylate. Then the cells were post-fixed in 1%

osmium tetroxide for 1 h and dehydrated through gradient

ethanol solutions (50%, 70%, 90% and 100%) and finally air dried

in hexamethyl disilazane (HMDS). The samples were gold

sputtered and then viewed with SEM.

2.6 Quantitative real-time PCR

HBMSCs were cultured in the powder extracts of Co-TCP or on

the Co-TCP ceramic discs. The samples were processed after 7

days of culture. Briefly, total RNA was extracted using TRIzol

reagent (Invitrogen) and 1 mg of total RNA was subjected to

reverse transcription into cDNA with SuperScript III (Invi-

trogen) in 20 mL reaction volume. 1 mL of cDNA from each

sample was used for each 25 mL real-time PCR reaction con-

taining the SYBR Green qPCR master mix (AB Applied Bio-

systems, Melbourne, Australia) and primers. The gene

expression of VEGF and 18S (housekeeping gene) was detected

by a real time-PCR machine (ABI Prism 7300, AB Applied

21688 | J. Mater. Chem., 2012, 22, 21686–21694

Biosystems). The relative expression of VEGF was normalized

against housekeeping gene 18S RNA. The primer sequences of

each gene were listed in Table 1.

For analysis of element concentrations, the culture media with

ceramic discs on day 7 were collected and the levels of Ca, Co and

P were measured using ICP-OES.

2.7 In vitro angiogenesis

An in vitro angiogenesis assay was conducted using ECMatrix�(Millipore, cat. no. ECM625), which consists of laminin,

collagen type IV, heparan sulfate proteoglycans, entactin and

nidogen. The powder extracts were prepared by soaking powders

in serum-free EBM-2 (Lonza) at a solid/liquid ratio of 200 mg

mL�1. After incubation at 37 �C for 24 h, the mixture was

centrifuged and the supernatant was collected and sterilized by

filtration through 0.2 mm filter membranes. The levels of Ca, P

and Co in the extracts were measured using ICP-OES.

Culture plates (96-well) were coated with ECMatrix�according to the manufacturer’s protocols. HUVECs (3 � 104

cells per well) were seeded on ECMatrix� with different powder

extracts supplemented with 1% FBS for 4, 8 and 18 h. At each

time point, five randomly selected fields of view were photo-

graphed per well under an inverted light microscope (Leica DMI

3000B). The number of branch points in HUVECs lines (node),

mesh-like circles (circle) and tube-like parallel cell lines (tube)

were quantified following the manufacturer’s instructions.

Nodes, circles and tubes are parameters of the gradual regener-

ation process of angiogenesis, representing the primary, interim

and later phases, respectively.23

2.8 Statistical analysis

All the data were expressed as means � standard deviation.

Statistical analysis was performed by the one-way ANOVA test.

A value of p < 0.05 was considered to be of statistical

significance.

3 Results

3.1 Characterization of Co-TCP powders

The Co-TCP powders were identified to be fully whitlockite (b-

TCP) phase through XRD analysis. The slight right shifts of the

XRD peaks, compared with the standard (JCPDS 09-0169), were

found in the patterns of Co2-TCP and Co5-TCP samples, but no

other phase was detected (Fig. 1). From the SEMmicrographs, it

could be seen that there was no obvious difference in

morphology and particle size of the powders (Fig. 2). As the EDS

results showed, the measured Co/(Ca + Co) ratios of Co-TCP

powders were close to the theoretical values (Fig. 2).

This journal is ª The Royal Society of Chemistry 2012

Fig. 1 XRD patterns of Co-TCP powders with different Co contents

calcined at 800 �C. All the three compositions were b-TCP phase.

Fig. 2 SEM micrographs of Co-TCP powders with different Co

contents together with EDS analysis. (a) b-TCP, (b) Co2-TCP and (c)

Co5-TCP.

Fig. 3 XRD patterns of the Co-TCP ceramics with different Co contents sinte

b-TCP phase; (b) 1100 �C, pure TCP had mixed a- and b-TCP phases, wher

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3.2 Characterization of Co-TCP ceramics

The XRD patterns of the Co-TCP ceramics with different Co

contents sintered at different temperatures are shown in Fig. 3.

When sintered at 1050 �C, the three ceramics were shown to be b-

TCP phase. However, when the temperature increased to

1100 �C, it was interesting to find that some peaks ascribed to the

a-phase appeared in the pattern of TCP ceramics without Co,

whereas the Co2-TCP and Co5-TCP ceramics still maintained

the b-phase.

SEM micrographs (Fig. 4) demonstrates that the surface

microstructure of the three ceramics sintered at different

temperatures. When sintered at 1050 �C, all the ceramics were

almost sintered, however, more micropores existed in the pure

TCP ceramics, compared to the Co-TCP ceramics. There was

no obvious difference in crystal morphology and size. When

sintered at 1100 �C, there were some melted crystals and micro-

cracks on the surface of pure TCP ceramics, however, the

phenomenon was not found in the Co2-TCP and Co5-TCP

ceramics. The grain sizes increased greatly with the increase of

the sintering temperature for all three ceramics. In addition,

micropores were barely visible in the Co2-TCP and Co5-TCP

ceramics.

Accordingly, the density test of the ceramics was performed

with the results plotted in Fig. 5. At 1050 �C there was no

significant difference among the three ceramics. With the

increase of the temperature to 1100 �C, the density of both Co2-

TCP and Co5-TCP ceramics increased, however, the density of

pure TCP sintered at 1100 and 1050 �C had no obvious differ-

ence. Interestingly, the density of the Co-TCP ceramics was

significantly higher than that of pure TCP when they were sin-

tered at 1100 �C.

3.3 Proliferation of HBMSCs cultured in the powder extracts

and on the ceramic discs of Co-TCP

HBMSCs were cultured both in the powder extracts and on the

ceramic discs. The results showed that cells cultured in all the

powder extracts presented a similar proliferation rate to that of

the blank control (Fig. 6a). Regarding the ceramic discs, all the

ceramics supported cell attachment and growth, showing

favourable cytocompatibility (Fig. 6b). There was no significant

difference for cell proliferation among pure TCP, Co2-TCP and

Co5-TCP in both powder extracts and disc forms.

red at different temperatures. (a) 1050 �C, all the three compositions were

eas Co incorporation stabilized the b-TCP phase.

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Fig. 4 SEM images of the surface microstructure of Co-TCP ceramics with different Co contents sintered at different temperatures. Scale bars¼ 10 mm.

Fig. 5 Density of the Co-TCP ceramics with different Co contents sin-

tered at different temperatures.

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The cell morphology seeded on the ceramics for 24 h is shown

in Fig. 7. Cells attached and spread on all the surfaces with

projected filopodia. No difference was noted in cell morphology

on all tested disc surfaces.

3.4 VEGF expression of HBMSCs

The VEGF expression of HBMSCs cultured with the Co-TCP

powder extracts or ceramic discs is shown in Fig. 8. The powder

extracts of Co5-TCP greatly upregulated the expression of

VEGF by 4-folds compared with that of pure TCP, while the

Co2-TCP powder extracts had no obvious effect on the

Fig. 6 Proliferation of HBMSCs cultured (a) in Co-TCP

21690 | J. Mater. Chem., 2012, 22, 21686–21694

expression of VEGF (Fig. 8a). When cells were cultured on the

ceramic discs, both the Co2-TCP and Co5-TCP ceramics

showed a stimulatory effect on the expression of VEGF as

compared to pure TCP sintered at the two temperatures (1050

and 1100 �C). The VEGF expression in HBMSCs of the Co2-

TCP and Co5-TCP ceramic discs showed no significant differ-

ence (Fig. 8b).

3.5 Element analysis of Ca, P and Co in the powder extracts

and ceramics conditioned media

The corresponding levels of Ca, P and Co in the powder extracts

and culture media with ceramic discs are listed in Table 2. For the

powder extracts, the release of Ca2+ ions decreased with the

increase of the Co substitution. The ion concentration of Co2+ in

the Co5-TCP powder extracts was about 12 mM, which was

much higher than that in the Co2-TCP powder extracts

(�1.5 mM). For the ceramic discs, both Co2-TCP and Co5-TCP

ceramics released a certain amount of Co2+ ions without signif-

icant difference between the different sintering temperatures.

Furthermore, the release of Co2+ ions increased with the increase

of Co content in Co-TCP. The release of Ca2+ ions decreased

with the increase of the Co content. Comparing the ceramics

sintered at 1100 to 1050 �C, the release of both Ca and P elements

decreased for the Co2-TCP and Co5-TCP ceramics. However,

pure TCP sintered at 1100 �C showed much higher levels of Ca

and P than that sintered at 1050 �C and Co-TCP.

powder extracts and (b) on Co-TCP ceramic discs.

This journal is ª The Royal Society of Chemistry 2012

Fig. 7 SEM micrographs of HBMSCs morphology seeded on Co-TCP ceramic discs for 24 h. Scale bars ¼ 50 mm.

Table 2 The levels of Ca, P and Co in the powder extracts and ceramicsconditioned media of DMEM (mM)

Co Ca P

Powder extracts TCP — 0.45 0.058Co2-TCP 1.5 � 10�3 0.43 0.14Co5-TCP 1.18 � 10�2 0.38 0.09

Ceramics 1050 �C TCP — 1.22 1.26Co2-TCP 4.6 � 10�3 0.96 1.00Co5-TCP 1.12 � 10�2 0.87 1.32

Ceramics 1100 �C TCP — 1.54 3.32Co2-TCP 4.9 � 10�3 0.94 0.94Co5-TCP 9.6 � 10�3 0.70 0.87

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3.6 In vitro angiogenesis

As shown in Fig. 9A, after 4 h, the HUVECs cultured in all the

compositions of powder extracts self-assembled and formed

branched nodes (node) and mesh-like circles (circle), the

phenomena of the primary and interim stages of angiogenesis. It

is worthy of note that the cells in the Co2-TCP extract formed

significantly more nodes and circles than the cells cultured in

pure TCP extract (Fig. 9A and B). Moreover, the Co2-TCP

extract induced the most tube-like parallel cell lines (tube), the

late phase of angiogenesis, among all the groups (Fig. 9A and B).

After 8 h of culture, the cells in the Co2-TCP extract showed

more nodes than the cells cultured in pure TCP and Co5-TCP

extracts (Fig. 9A and C). In addition, Co2-TCP still kept the

advantage over TCP in stimulating the formation of circles

(Fig. 9A and C). According to the instructions for the angio-

genesis assay kit, HUVECs should begin to undergo apoptosis

after 18 h of culture, which was the case in all the groups

(Fig. 9A). However, the cells in the Co2-TCP extract maintained

a better network structure than other groups (Fig. 9A and D).

These results suggested that Co-TCP could promote the angio-

genic capacity due to the release of Co2+ ions, and the effect was

Co2+ ion concentration-dependent, as shown in Table 3.

4 Discussion

In the present study, Co-substituted b-TCP ceramics have been

successfully prepared by replacing different amounts of Ca. It is

Fig. 8 VEGF expression of HBMSCs cultured (a) in the Co-TCP p

This journal is ª The Royal Society of Chemistry 2012

interesting to find that the incorporation of Co into b-TCP

ceramics stabilized the b phase and enhanced their sinterability.

Both Co-TCP powder extracts and ceramic discs supported the

proliferation of HBMSCs and most importantly, stimulated the

expression of VEGF through the release of Co2+ ions as

compared to pure TCP. The release of Co2+ ions could be

controlled by controlling the content of Co in TCP. Further-

more, Co-TCP showed higher in vitro angiogenic potential than

pure TCP. It is suggested that the incorporation of Co into

biomaterials, such as b-TCP, is a viable way to enhance the

potential angiogenic property of biomaterials. Co-TCP ceramics

are promising to be used as bone graft substitutes with poten-

tially improved angiogenesis.

owder extracts and (b) on the Co-TCP ceramic discs for 7 days.

J. Mater. Chem., 2012, 22, 21686–21694 | 21691

Fig. 9 In vitro angiogenesis of HUVECs cultured on ECMatrix� in the presence of Co-TCP extracts. (A) The optical photos of HUVECs cultured on

ECMatrix� in the presence of powder extracts for 4, 8 and 18 h. (B–D) The statistics of the number of formed nodes, circles and tubes after culture for 4,

8 and 18 h, respectively. Data represent means � SD (n ¼ 4). *p < 0.05.

Table 3 The levels of Ca, P and Co in the powder extracts prepared byEBM-2 (mM)

Co Ca P

TCP — 2.29 0.07Co2-TCP 1.8 � 10�3 2.65 0.10Co5-TCP 7.5 � 10�3 2.12 0.08

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One of the interesting results is that the incorporation of Co

into b-TCP ceramics stabilized their b phase. There are two

possible reasons to explain this result. Firstly, as Co2+ ions have

a smaller ionic radius (0.074 nm) than Ca2+ ions (0.099 nm), the

substitution of Ca with Co resulted in a slightly right shift of the

XRD characteristic peaks, which indicates a reduction of

interplanar distance and the crystal unit volume. Previously, it

was found that the incorporation of Sr into CaSiO3 (CS)

stimulated the phase transition of CS from b to a due to the

larger ionic radius of Sr2+ than Ca2+.24 Therefore, it is specu-

lated that the smaller interplanar distance and crystal volume of

Co-TCP inhibited the transport of atoms and further sup-

pressed the phase transition. Secondly, previous studies have

shown that the incorporation of Mg and Zn can increase the

phase transformation temperature and stabilize the b-TCP

21692 | J. Mater. Chem., 2012, 22, 21686–21694

structure.25–28 Mg and Zn are found to preferentially occupy the

Ca(5) site and the bond distances of Mg(5)–O (2.070–2.084 �A)

and Zn(5)–O (2.175–2.185 �A) were shorter than the Ca(5)–O

distance (2.238–2.287 �A), showing a more ideal octahedral

geometry.29,30 The ionic radius of Co2+ is similar to Zn2+ (0.074

nm), which suggests that Co can also reside in the Ca(5) site,

known as the Mg sites, which accommodate divalent cations

with ionic radius from 0.060 to 0.080 nm.31 The Co(II)–O bond

distance was reported to be 2.093 �A (coordination

number ¼ 6),32 shorter than the Ca–O bond distance.

Furthermore, Brown and Shannon reported that these octahe-

dral sites containing Co2+ were generally less distorted than

Ca2+.33 Thus, the more ideal octahedral structure could be

another reason for the stabilized effects of Co substitution on

the phase transition of b-TCP.

In our study, the incorporation of Co into b-TCP increased the

density of the sintered ceramics. Previous studies have shown

that the phase transformation of b- to a-TCP is closely related

with the expansion of sample volume and declining shrinkage

rate, which prevents TCP from further densification.34,35 In this

study, Co substitution inhibited the phase transition of b-TCP,

making it further enhance the bulk density when sintered at a

higher temperature (1100 �C). However, for the pure TCP

ceramics, the part phase transformation from the b to a phase

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may be the main reason for the inhibition of their densification

even at the higher temperature of 1100 �C. A previous study has

found that CoO could be used as a sintering additive to enhance

the densification rate of Ce0.8Gd0.2O1.9 ceramics.36 It is specu-

lated that in this study, Co could be acting as a sintering aid to

enhance the sinterability of the TCP ceramics.

It is well-known that b-TCP has excellent biocompatibility to

support cell attachment and proliferation. Our results showed

that the incorporation of Co into b-TCP had no obvious effect

on the cell viability, indicating that Co-TCP had a comparable

cytocompatibility with pure TCP. Previous studies have shown

that Co2+ ions with high concentrations (over 100 mM) may

cause cytotoxicity.37,38 In our study, the concentration of Co2+

ions released from the Co2-TCP and Co5-TCP powders and the

ceramic discs was no more than 10 mM, a relatively low

concentration, which would not bring the potential toxicity.

Moreover, Co-TCP showed a significantly higher VEGF

expression level of HBMSCs than pure TCP. VEGF plays a

critical role in the development and progression of the angio-

genesis system both in normal physiological and pathological

conditions.39 It is regarded as one of the key factors coupling

vascularization and osteogenesis.40 As the three ceramics have

similar surface microstructures at the same temperature, it is

speculated that the release of Co2+ ions from the Co-TCP

ceramics mainly contributes to the improved VEGF expression

of HBMSCs. Previous studies have shown that Co2+ ions play an

important role in inducing the hypoxia effect and further

enhancing VEGF expression of cells.41–43 Our study has shown

that the Co2+ ions released from the Co-TCP materials, with a

concentration range of about 5–10 mM, enhanced the gene

expression of VEGF. These results indicated that Co-TCP might

possess the ability to enhance the angiogenic potential more than

pure TCP.

It has been demonstrated that the migration of ECs and the

formation of tube-like network structures, called capillary cords,

are critical steps for angiogenesis.44,45 ECs are widely used in the

model for the angiogenic property test of biomaterials.46,47 In this

study, HUVECs were employed to investigate whether the

incorporation of Co into b-TCP could enhance its angiogenic

property. The results did show that the Co-TCP powder extracts

stimulated the formation of a network structure of HUVECs as

compared with pure TCP, and the stimulating effect could be

modulated by tailoring the content of Co substitution, which

further predicted that the incorporation of a certain amount of

Co into biomaterials could bring higher angiogenic capacity.

Comparing the effects of the Co-TCP powder extracts on VEGF

expression and in vitro angiogenesis, it could be found that

although the increased VEGF expression of HBMSCs in Co2-

TCP was not more significant than that in the TCP extract, the

capillary-like network formation of HUVECs in the extract of

Co2-TCP was enhanced. For Co5-TCP, a significant increase in

VEGF expression in HBMSCs was detected, however, the

enhanced network structure formation by HUVECs in

the matrix gel was marginal in the Co5-TCP extract compared to

the TCP control, indicating a need to optimize the content of Co

substitution in the TCP materials. Therefore, future study will be

conducted to find out the optimum content of Co substitution

and the controllable release of Co2+ ions from materials for the

desirable angiogenesis.

This journal is ª The Royal Society of Chemistry 2012

5 Conclusions

Co-substituted b-TCP has been successfully prepared. The

incorporation of Co into b-TCP stabilized the b-phase structure

of b-TCP and improved the sinterability. Co-TCP showed

excellent cytocompatibility to support the growth of HBMSCs in

both powder extract and ceramic disc forms. Most importantly,

the Co2+ ions released from the Co-TCP powders and ceramic

discs significantly upregulated the VEGF expression of HBMSCs

as compared to pure TCP, and the release of Co2+ ions can be

controlled by different Co substitution contents. Moreover, Co-

TCP showed an improved in vitro angiogenic potential as

compared with pure b-TCP. Our results have suggested that it is

promising to incorporate Co into biomaterials to promote

VEGF expression of bone-forming cells and further enhance

angiogenic capacity. Co-TCP may be used for bone tissue engi-

neering applications with an improved angiogenic potential.

Acknowledgements

This work is financially supported by National Natural Science

Foundation of China (grant no. 81190132), Australian Research

Council Discovery Project (ARC DP120103697) and the Prince

Charles Hospital Research Foundation (TCPHMS2011-05). We

appreciate the technical support of Mr Tony Raftery (X-ray

Analysis Facility, Queensland University of Technology) for

XRD andMr Shane Russel (Faculty of Science and Technology,

Queensland University of Technology) for ICP-OES.

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